Screening Metagenomic Libraries

Recently, metagenomic library screening provided a few more nitrilases, whose genes were obtained from clones growing on cinnamonitrile or a nitrile mixture. One of the enzymes was selected for the monohydrolysis of 2-methylglutaronitrile into 

4- cyanopentanoic acid, a precursor of l,5-dimethyl-2-piperidone [14]. A different method based on PCR was used to screen metagenomic DNA for new nitrile hydratases and resulted in the isolation of six active enzymes [15]. 

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An effective and generic method of screening of BVMO activity is indispensable for screening metagenome libraries as these libraries are often in the range of 100000-1000000 clones. Therefore, it is a challenge to develop a novel way to efficiently detect BVMO activity in gene libraries. This would facilitate... 

But there is still another point, not yet discussed but with considerable potential, which may also impact eventually on technical asymmetric catalysis. Even though biocatalysts are efficient, active, and selective, there still remains one big disadvantage At present, there is not yet an appropriate enzyme known or available for every given chemical reaction. It is estimated that about 25 000 enzymes exist in Nature, and 90% of these have still to be discovered [28, 29]. New biocatalysts are made available nowadays not only from screening known organism but also via screening metagenomic libraries and directed evolution techniques [30]. 

Functional screening of a soil metagenomic library for cellulases revealed a total of eight cellulolytic clones, one of which was purified and characterized [58]. Despite the fact that this library had been generated from a soil sample collected from a... 

A number of metagenomic libraries hosted in E. coli have now been screened for clones that produce either antibacterial activities or color. In addition to hits that are unique to each library, these studies have encountered four common hits. These include the antibacterially active long-chain A-acyl amino acid-producing clones described earlier in this section, red antibacterially active clones that express aminolevulinic acid synthases (hemA), brown clones that produce melanin-like polymers, and blue clones that produce mixtures of indigo (25) and indinibin... 

In search of alternative thiamine pathways that might be more susceptible to metabolic pathway engineering, metagenomic libraries from various habitats were screened using a thiamine auxotrophic, tAiC-deleted E. coli screening host. In addition, the host contained a deletion in an early pur gene to prevent the supply of AIR, the ThiC substrate, to remove those clones which are complemented by the conventional ThiC dependent pathway. None of the hits encoded a genuinely novel thiamine pathway (BRAIN AG, unpublished). 

The screening of a metagenomic library derived from loam soil afforded a novel two-component SMO, which has been heterologously expressed in E. coli [52], This biocatalytic system was able to perform selective sulfoxidations of alkyl aryl sulfides, providing the recovered (R)-sulfoxides with good to high selectivities and moderate conversions after 16h. Ethyl phenyl sulfide was the best substrate for this enzyme, as the final sulfoxide was obtained with 92% ee. It therefore represents an interesting SMO alternative. 

DeSantis et have reported the discovery of new nitrilases through the screening of genomic libraries created by the extraction of DNA from various environments (metagenomics). In preliminary experiments, using 25 mM mandelonitrile in pH 8 buffer containing 10% methanol and 0.12 g mL of one of these nitrilases, the acid was produced quantitatively with 98 % ee within 10 min. The product was subsequently shown to be (7 )-mandelic acid after isolation in 86 % yield. In a parallel reaction, (/ )-2-chloromande-lic acid was produced at a seventeenth of the rate (Scheme 1.44). 

Figure 2.3 Metagenomic cloning experiments. Isolation of genomic DNA directly from environments (soil, plants, mixed environments or thermal-vent worms are the examples Illustrated here) can recover DNA fragments which could encode for enzymes. The DNA fragments can be ligated to plasmids or DNA linkers, and then subjected to functional screening (expression cloning) and/or sequence analysis. Amplification by PCR can sometimes be used to yield libraries enriched with clones containing selected sequence motifs relating to families of enzymes...

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